Why we get sick or age quicker as adults may be linked to chemical exposure early in development... not just our own time in the womb but also that of several generations before us.

This astonishing result builds on research first reported in June 2005, in which Anway et al. found through studies with rats that a developmental exposure can decrease sperm count, not just in the exposed offspring but in at least three subsequent generations as well. In human terms, this would mean that an exposure experienced by your great-great-grandmother while your great-grandfather was in the womb had an adverse effect on your sperm count.

Most remarkably, the effects were not transmitted by mutations, or changes in DNA sequence. Instead, they appeared to be the result of altered methylation patterns: changes in molecules attached to DNA that control whether a gene can be turned on.

Anway et al.'s results surprised toxicologists and evolutionary biologists alike because they demonstrated inheritance of an acquired characteristic that was not mediated by mutations, but instead through epigenetic inheritance.

Now from the same lab comes a new pair of studies showing chronic diseases of ageing can be transmitted across generations in the same way. The first, by Anway et al., reports that tumors, including male breast cancer, prostate and kidney disease and testis problems increase in subsequent generations following exposure at a key time in development. The second, by Chang et al., identifies a series of genes whose molecular state is altered by the exposures and demonstrates that the changes are transmitted epigenetically. They also report that the affected genes are associated with several human diseases, including Alzheimer's.

What is epigenetic inheritance?

In genetic inheritance, traits are passed from one generation to the next via DNA sequences in genes. Differences in a DNA sequence specify differences in a trait.

Epigenetic inheritance involves passing a trait from one generation to the next without a difference in DNA sequence. Known mechanisms of epigenetic inheritance include changes in molecular structures around the DNA so that while the gene is the same, the gene behaves differently. For example, genes switch on and off in response to hormonal signals. Changes in molecular conformation around the gene can prevent that from happening. This can change developmental processes, alter disease resistance, etc.

In all, 85% of the experimental animals developed some disease. Many of the exposed animals in each of the generations studied had multiple problems. The affects on the prostate, kidney, breast, and testis were similar from generation to generation. In fact, those directly exposed in the womb were often less affected than subsequent generations.

These studies are important because they reveal another way that adult diseases can originate. If confirmed at environmentally-relevant doses, they represent a severe challenge to epidemiology as it is currently practiced.

How could this work?

The researchers exposed the pregnant rats at a very specific time early in development during the period just prior to when the fetus's sex is being determined. At this time, DNA methylation is removed temporarily, and then re-established in a way that is specific to males vs. females. Crucially, the cells that ultimately are going to become sperm and eggs in the next generation (the primary germ cells) are de-methylated and re-methylated also. Exposure at this time appears to permanently alter methylation in the primary germ cells, which means the changes are passed to the next generation.

Most epidemiology has focused on the role that adult exposures may play in adult diseases, for example, breast cancer. A much smaller number of studies have attempted to examine how fetal exposures affect adult health, for example, testicular cancer. And a very small number of studies have examined the health of daughters of women exposed to diethylstilbestrol in the womb, for example, finding menstrual irregularities. None have looked for the sorts of transgenerational impacts reported by Anway et al. as a result of exposure to environmental contaminants.

An important limitation to this work is that it used relatively high levels--amounts unlikely to be encountered by most people-- of vinclozolin, an endocrine-disrupting fungicide, to induce the effect, . Hence while it does confirm that multi-generational transmission through epigenetic changes can occur, it doesn't indicate how likely these effects are to be observed in people.

What did they do?

In the first study, Anway et al. injected pregnant rats with the fungicide vinclozolin, an anti-androgenic compound, during a sensitive period of embryonic gonad development and then examined the incident of disease in the next four generations of adult rats.

In the experiment, six pregnant rats (the F0 generation) were injected daily with 100 mg/kg/day vinclozolin from 8 to 14 days after fertilization. Only the mothers and the developing first generation were directly exposed to the anti-androgen.

As in a previous study, the researchers picked the time frame because fundamental changes in gonad development occur during this period. Cells that ultimately become the rat’s sperm, called primordial germ cells, migrate into position in the embryo. As they are migrating, a chemical transformation takes place that removes DNA methylation. The methylation pattern is then re-instituted when the sex of the embryo’s gonads is set through genetic signaling.

To determine how the fungicide influences health and aging from generation to generation, the offspring were bred for four generations. Mature, males from injected rats, known as the F1 generation, were bred with mature F1 females from other injected mothers producing F2 grandchildren. Then, F2 males and F2 females were mated producing F3 offspring, and F3 males and F3 females mated producing the F4 generation.

No inbreeding or family crosses were made. Offspring of control rats injected with a similar solution but without the fungicide were mated in the same way.

Next, researchers identified diseases and other abnormalities in grown vinclozolin and control rats and compared the types and frequencies among generations. To do this, adult rats of both sexes from the F1, F2, F3, and F4 generations were collected at 6 to 14 months old. The animals and many tissues were examined for tumors, abnormal cells, immune problems (inflammation, infection), and premature aging (abnormal hair color, reduced mobility, and weight loss). Blood analysis included red and white cell counts, metabolic products, and testosterone.

Anway et al. also bred F2 vinclozolin males to non-control females of the same strain (wild type). Similarly, F2 vinclozolin females were bred to wild-type non-control males. These outcross experiments determine if effects are transmitted through male or female line.

In the second study, Chang et al. compared methylation patterns in DNA extracted from sperm of multiple generations of treated vs. control animals, from F1 to F3. As in Anway et al., the only exposure was during the first generation (pregnant female=F0). Subsequent generations were generated by breeding males from an exposed lineage with females from an exposed lineage, while avoiding inbreeding. They also examined levels of gene expression of the genes with altered methylation patterns in different tissues.

What did they find?

Up to 85% of vinclozolin experimental animals developed diseases and tissue abnormalities that persisted through the four generations. Both males and females were affected, and many animals had more than one problem. The main afflictions were tumors, prostate and kidney disease, testis abnormalities and immune problems.

Overall, exposed animals had higher incidence of diseased or abnormal animals than in controls.

Animals in the vinclozolin- treated lineage had higher incidence of diseases than controls. Figure to right pools generations F1 to F4.

Adapted from Anway et al.

Between 12 and 33% of F1-F4 generation adult rats developed breast, lung and skin tumors. Less than half of those were malignant. Controls, on the other hand, were tumor free in all generations

Prostate disease affected about half of all vinclozolin males in all generations. The 45 to 55% of males with lesions or shrunken or inflamed tissue is more than double the rate found in the control males.

Both sexes developed kidney diseases that were sometimes detected through increased waste products in blood samples. Up to 50% of males and 30% females had tissue impairments.

As in a previous study, males had increased spermatogenic cell apoptosis, sperm defects, and low sperm counts when compared to control males across all generations. In this study, 13 – 38% of vinclozolin males were affected.

In several types of disease states, the F1 generation had lower prevalence than later generations.

Graph to left summarizes incidence of different disease states in each generation of exposed animal.

Adapted from Anway et al.

Several observed differences need further investigation, according to Anway et al.

For instance, inner ear and respiratory infections and other types of inflammation affected 12 to 33% of vinclozolin rats but no control animals. As yet unexplained are other immune abnormalities and a dramatic increase in cholesterol in the 6 – 14 month old vinclozolin rats.

They also observed signs of premature ageing in treated animals. Characteristics, such as decreased grooming and movement, increased skin abnormalities, and periodic weight loss were seen in half of all F1 – F4 animals although these tendencies are not seen in control animals until after 18 months. Sick animals share the same characteristics so further research is needed to distinguish ageing from disease.

In the methylation comparisons, initial screening for methylation differences identified 25 candidate genes for study. Further analysis showed that 15 were definitely hypermethylated in the vinclozolin lineage compared to controls.

In F1 and F2 they further tested for changes in expression of the genes that were hypermethylated, examining gene expression in the fetal testes of exposed vs. control lineages. Several genes had reduced expression while others had increased.

One notable case was the gene NCAMI, which is active in the brain. They therefore compared activity of NCAMI in F3 control vs. F3 treated lineages and found that expression of NCAMI was suppressed by over 10-fold in F3 treated animals. Thus they were able to confirm that vinclozolin treatment in F0 led to altered methylation patterns in sperm of subsequent generations and reduction of expression in the brain of F3 animals, consistent with their proposal that epigenetic alterations in the germ line leads to differences in gene expression in tissues.

Chang et al. reviewed prior literature on possible links of the genes with altered methylation patterns to human disease conditions. An number of them were associated with diseases, including Alzheimer's, schizophrenia and infertility. "Interestingly, all of these genes have been shown to have an epigenetic component to the disease and/or gene identified."

Table adapted from Chang et al.; references available in Chang et al.

What does it mean?

According to Anway et al., their new research shows that “in utero exposure to an environmental compound, the endocrine disruptor vinclozolin, has the ability to induce multiple transgenerational disease states.”

If the mechanism of epigenetic inheritance of disease states they have discovered operates at environmentally-relevant levels, their findings reveal a profound blind-spot in epidemiology and toxicology.

Two aspects of this work are especially important. The first--that adult chronic disease states can have contaminant-induced origins in fetal development--is consistent with an emerging body of scientific research on the fetal origins of adult disease. As noted above, few epidemiological studies are designed to test causal mechanisms like this, despite an abundance of animal data consistent with this pattern (for example).

Anway et al.'s work broadens the range of adult disease states that are now tied to fetal origins, but it is a logical extension of that literature. Chang et al.'s demonstration that altered methylation predicts altered gene expression in both fetal and adult tissue, in genes relevant to human disease, is an important piece of evidence that adds mechanistic details to the overall picture.

The second key aspect of their work-- that contaminant-induced disease states can be inherited for multiple generations through epigenetic mechanisms-- represents a deeper and even more daunting challenge, to toxicology, epidemiology and indeed to evolutionary biology. If confirmed to work in people and to operate at environmentally-relevant levels of exposure, it will require radical changes in how toxicological and epidemiological studies are conducted, and in how public health standards are established.

Until their publication last year, the only hint of such a possibility was from work on DES, noted above, showing in animals and people that F2 was affected adversely by DES delivered to F0 with F1 in utero. No mechanistic details at the molecular level have been offered to explain that F2 effect, although DES is known to alter methylation patterns in F1. It has been plausible to hypothesize either (1) F2 was affected directly through exposure experienced by F1's primordial germ cells (already formed by the time of DES exposure); or (2) that hormonal changes in F1 caused by direct DES exposure then adversely affected F2. Effects shown by Anway et al. and Chang et al. on F3 and F4 eliminate those as plausible explanations in this case.

This new pair of papers confirms their earlier publication and extends the findings from one adverse effect (lowered sperm count) to a range of adult-onset diseases; it also adds more detail about methylation and gene expression, and reveals that some of the affected genes have been tied previously to human diseases relevant to observed changes in their rats.

Anway et al. offer the following additional perspective their findings:

"As a comparison the frequencies [of disease states] observed are similar to that seen in the human population. Prostatic lesions occur in 50% of men over the age of 50 years, as compared to the 51% observed in the current study. The progression of human prostatic disease has been suggested to involve an initial atrophy of epithelium and glands followed by prostititis, as observed in the current study. Renal lesions occur at frequencies in specific human subpopulations similar to the 30% observed in the current study. The abnormal kidney morphology observed corresponded to changes in serum BUN and creatinine levels, as is seen in the human population. Testis abnormalities occur in approximately 10-15% of the human male population compared to the 30% prevalence observed in the current study. The morphological changes and spermatogenic cell defects are similar to the reported human defects. The tumor rates for breast cancer are approximately 15% in the human population, but less than 1% in males. In contrast, the male rats in the F1-F4 vinclozolin generations had approximately a 10% frequency. As with human tumors, rat tumors observed were primarily of epithelial cell origin with a low frequency of metastasis. Overall, several similarities in frequency and etiology were made with the abnormalities observed in the current study with those found in humans. Future studies are now required to allow a comparison of the rat observations to human disease."

Is their finding unique to vinclozolin? That seems unlikely. Anway et al. had shown previously that methoxychlor, a pesticide, causes epigenetically-inherited changes in sperm count reductions, so the basic mechanism is not unique to vinclozolin. Vinclozolin is one of many compounds known to have anti-androgenic activities; methoxychlor is one of many with estrogenic properties. Both these endocrine disruptors alter gene expression. Vinclozolin's known effects on rodents resemble those of other anti-androgenic compounds, like some of the phthalates. Many endocrine disruptors can alter gene expression via changes in DNA methylation, for example bisphenol A. Therefore there is no a priori reason to conclude that vinclozolin is unique. It will be important to test this with a range of endocrine disrupting compounds.

Key questions:

Which adult-onset health conditions in people result from the fetal environment?

Are human diseases caused by environmental exposures transferred from one generation to the next via epigenetic mechanisms?

Do other endocrine-disrupting compounds also affect epigenetic inheritance, especially compounds, like bisphenol A, that alter gene expression at much lower, environmentally-relevant levels than vinclozolin.